Prephenate dehydratase is a regulatory enzyme in the pathway for biosynthesis of phenylalanine in Bacillus subtilis. It is activated by methionine and leucine which cause the monomer to associate to a tetramer and inhibited by phenylalanine and tryptophan, which associates it to the monomer. We plan to investigate the kinetics and nature of the aggregation using physical methods. In addition, through chemical modification and affinity labeling studies we will attempt to gain insight into the chemical basis for the aggregation and the catalytic mechanism for the enzyme. In order to do chemical and physical studies, pure enzyme is required. We will have to finish up work on the purification that is being approached in two ways. One is to take advantage of the large difference in size between the monomer (approximately 50,000 daltons) and the tetramer (approximately 200,000 daltons) by using gel filtration to purify the enzyme to homogeneity. The other way is to design appropriate affinity chromatographic systems based on the structures of the substrate and the effectors.